Congcong Xu

Circular RNAs (circRNAs) are covalently closed RNA molecules with exceptional intrinsic stability and when properly designed, purified, nucleoside-modified, and delivered, show reduced unwanted innate immune activation, endowing them with unique advantages for vaccines, cancer immunotherapy, and protein replacement therapies. Synthetic circRNAs can be generated via enzymatic ligation, chemical ligation, or ribozyme-mediated circularization; however, efficient translation and scalable manufacturing remain major bottlenecks. In this Review, we survey current production strategies and therapeutic applications of synthetic circRNAs, and delineate engineering approaches that enhance molecular stability through nucleoside modification and sequence optimization, as well as strategies to improve translational efficiency, including internal ribosome entry site (IRES) and cap-mimicking elements, epitranscriptomic modifications, and rational sequence design. We further examine how these optimizations intersect with immunogenicity and process development, highlighting the critical quality attributes that govern circRNA product consistency and performance. Finally, we discuss emerging opportunities for algorithm-driven design and integrated engineering platforms, emphasizing their potential to accelerate the clinical translation of circRNA therapeutics.

Synthetic circular mRNA (hereafter referred to as circRNA) reduces susceptibility to exonuclease‐mediated degradation by its covalently closed circular structure, enabling prolonged protein expression for therapeutic applications. In this circular format, protein expression from engineered circRNAs is achieved mainly through cap‐independent translation initiation, commonly mediated by internal ribosome entry site (IRES) elements whose activity is influenced by RNA structure. Consequently, the coding sequence (CDS) and other elements should be designed with consideration of inter‐region base pairing that can shift IRES folding, a constraint not explicitly addressed by existing linear mRNA CDS optimization algorithms. Here, we present circ Design, an algorithm that explicitly incorporates IRES structural deviation into circRNA sequence design while jointly optimizing codon adaptation and thermodynamic stability. In a rabies virus glycoprotein (RABV‐G) vaccine model, circ Design‐generated circRNAs showed improved stability, translation efficiency, and vaccine immunogenicity compared with benchmark sequences optimized using conventional linear mRNA CDS design strategies, with CR3 achieving a 3.5‐fold increase in neutralizing antibody titers. Polysome profiling and targeted IRES‐disruption experiments support IRES structural integrity as a critical determinant of circRNA translation performance. Together, these results establish IRES structural preservation as a mechanistic design principle for circRNA engineering and position circ Design as a rational framework for therapeutic circRNA development.

Synthetic circular mRNA (hereafter referred to as circRNA) reduces susceptibility to exonuclease‐mediated degradation by its covalently closed circular structure, enabling prolonged protein expression for therapeutic applications. In this circular format, protein expression from engineered circRNAs is achieved mainly through cap‐independent translation initiation, commonly mediated by internal ribosome entry site (IRES) elements whose activity is influenced by RNA structure. Consequently, the coding sequence (CDS) and other elements should be designed with consideration of inter‐region base pairing that can shift IRES folding, a constraint not explicitly addressed by existing linear mRNA CDS optimization algorithms. Here, we present circ Design, an algorithm that explicitly incorporates IRES structural deviation into circRNA sequence design while jointly optimizing codon adaptation and thermodynamic stability. In a rabies virus glycoprotein (RABV‐G) vaccine model, circ Design‐generated circRNAs showed improved stability, translation efficiency, and vaccine immunogenicity compared with benchmark sequences optimized using conventional linear mRNA CDS design strategies, with CR3 achieving a 3.5‐fold increase in neutralizing antibody titers. Polysome profiling and targeted IRES‐disruption experiments support IRES structural integrity as a critical determinant of circRNA translation performance. Together, these results establish IRES structural preservation as a mechanistic design principle for circRNA engineering and position circ Design as a rational framework for therapeutic circRNA development.

Lymph nodes are the primary sites where adaptive immunity is initiated, yet most messenger RNA cancer vaccines reach them inefficiently and instead accumulate in organs such as the liver, limiting therapeutic potency and increasing systemic toxicity. Here we developed a transferrin receptor-associating polyplex formed by electrostatic complexation of mRNA with low-molecular-weight polyethylenimine that had been chemically modified with cyclic disulfide monomers to enhance nucleic acid binding stability, enable thiol-based transferrin receptor engagement and reduce off-target liver uptake. After subcutaneous administration, these polyplexes activated innate immunity, rapidly recruited monocytes with high transferrin receptor expression and bound these cells through cyclic disulfide-mediated interactions. Monocytes then trafficked the vaccine to draining lymph nodes, where mRNA translation and antigen presentation occurred. Delivery of ovalbumin and interleukin 12 mRNA elicited strong antigen-specific cytotoxic T cell responses and inhibited melanoma progression and metastatic disease. Studies using Survivin and human papillomavirus antigens in distinct tumour models demonstrated broad applicability. This monocyte-driven lymph node-targeting strategy enables potent and selective delivery of mRNA cancer vaccines.